Carp haemoglobin iron‐imidazole linkage and subunit equivalence; evidence from resonance Raman spectroscopy of deoxy‐ and nitrosyl forms

Nitrosyl hemoglobin (Hb) from carp shows a resonance Raman spectrum characteristic of 6‐coordinate (NO) heme. Switching the quaternary structure from R to T by addition of insitol hexaphosphate (IHP) at pH 5.8 does not produce a 5‐coordinate spectrum, as it does for human Hb. Only intensity changes are seen for bands at ∼490, 387 and 357 cm⁻¹, indicating a subtle alteration in the porphyrin‐protein interaction. In carp deoxy Hb, IHP addition produced a shift of the Fe‐ImH (ImH = imidazole) stretching mode, from 223 to 214 cm⁻¹. The band remained symmetrical, indicating essential equivalence of the subunits. This is in contrast to human deoxy Hb for which the R‐T shift in the Fe‐ImH frequency is much greater in the α than the β chains. In carp Hb all the Fe‐ImH bonds appear to be weakened in the T state, but to an extent insufficient to induce rupture upon binding NO.